The studies in this Section have led to the hypothesis that in proteins exists some new delocalized interaction that is mediated by a closed loop of contacting groups formed after folding and generates extra energy to stabilize the structure. As described in another report A. Fisher and H. Taniuchi have located four hypothetical closed loops in cytochrome c. Of these four, closed loop 1 is located above the heme and forms first in fragment complexation, closed loop 2 located at the left side of the heme is assumed to order residues 28 to 38 and closed loop 3 located at the right side to stabilize the Met 80-S-Fe bond. The previous studies have indicated that closed loop 2 interacts with closed loop 3. To analyze thermodynamics of this closed loop 2-closed loop 3 interaction we use the three fragment complex ferro(1- 25)H.(28-38).(39-104) and a fragment exchange technique. The previous studies have shown that in the presence of excess of fragment (28-38) it is possible to measure the rate of direct dissociation of fragment (39-104) i.e. without going through two fragment complex (125)H.(39-104). On the basis of this principle we plan to measure the effect of substitution of leucine 32 and leucine 35 with norvaline (one at a time) on the binding force of fragment (39-104). Thus, we have prepared the heme- and apofragments and radiolabelled (39-104) determined the dissociation rate of complex ferro(1-25)H.(39-104) as a function of temperature, resulting in activation Gibbs energy, 18.25 kcal/mol at 25 degrees C; activation enthalpy, 52.8 within 5.6 kcal/mol; and activation entropy, 116 within 20eu. This combined with the previous data suggest that reduction of heme strengthens the interaction of closed loop 1 or closed loop 3 or both. Using this information and the previous data of dissociation constants, measurements of the dissociation rate of fragment (39104) from complex ferro-(1- 25)H.(28-38).(39-104) as a function of concentration of free fragment (28-38) should allow us to determine the rate constant of the direct dissociation. The same procedure will be used for the complex containing substitution at position 32 or 35.